Virus-like particles comprising a matrix protein from a plant enveloped virus and uses thereof

ABSTRACT

The present invention relates to novel virus-like particles (VLPs) comprising a matrix protein derived from a first plant enveloped virus and a surface polypeptide. The surface polypeptide comprises (a) a surface exposed portion derived from a target polypeptide (b) a transmembrane domain, and (c) a cytosolic tail derived from a transmembrane (e.g., glycoprotein) of a second plant enveloped virus. The target polypeptide may be antigenic or therapeutic. The first and the second plant enveloped viruses may be the same. Either plant enveloped virus may be a plant rhabdovirus. Also provided are methods of making and using the VLPs.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a U.S. national phase application of International Application No. PCT/US2013/032096, filed Mar. 15, 2013 claiming the benefit of U.S. Provisional Application No. 61/614,141, filed Mar. 22, 2012, the content of each of which is incorporated herein by its entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates generally to enveloped virus-like particles (VLPs) preferably produced in plants, and methods for making and using the VLPs.

BACKGROUND OF THE INVENTION

Virus-like particles (VLPs) are complex structures formed by self-assembling viral proteins without the presence of the viral genome. The VLP platform is increasingly used to enhance target-specific immunogenicity. VLPs can be produced in several expression systems, including plants. Production of VLPs in plants offers additional advantages, including safety and time efficiency. However, formation of VLPs in plants is part of a physiological process and results in formation of particles of various sizes and shapes, making commercial manufacturing and a smooth regulatory path highly challenging. The size of wild-type influenza virus particles depends on the strain and varies between 70 and 270 nm (Nayak et al., 2009, Virus Res 143(2):147-161). Plant-produced influenza viral particles (VLPs) have different shapes and a broad size range. For example, enveloped pleiomorphic influenza VLPs have been reported to be 100 to 150 nm in size. (Vezina et al., 2011, BioPharm International Supplements 24(5):s27-s30). Thus, there remains a need for more uniform VLPs suitable vaccine development.

SUMMARY OF THE INVENTION

The disclosed subject matter of the present invention relates to novel virus-like particles (VLPs) having a matrix protein derived from an enveloped virus and a surface polypeptide and their uses. These VLPs are enveloped, and substantially uniform in size.

According to one aspect of the present invention, a virus-like particle (VLP) is provided. The VLP comprises a matrix protein derived from a first plant enveloped virus and a surface polypeptide. The surface polypeptide comprises a surface exposed portion derived from a target polypeptide, a transmembrane domain, and a cytosolic tail. The cytosolic tail is derived from a transmembrane protein (e.g., glycoprotein) of a second plant enveloped virus. The VLP may be produced in a plant cell, a plant, or a portion of a plant.

The first and the second plant enveloped viruses may be the same or different, preferably the same. Either may be a plant rhabdovirus or non-rhabdovirus. Preferably, the first plant enveloped virus is a plant rhabdovirus. The plant rhabdovirus may be selected from the group consisting of Lettuce Necrotic Yellows virus (LNYV), Northern Cereal Mosaic virus (NCMV), Sonhus Virus (SonV) and Broccoli necrotic yellows virus (BNYV). Preferably, the plant rhabdovirus is Lettuce Necrotic Yellows virus (LNYV).

The surface exposed portion of the surface polypeptide may be an antigenic polypeptide, such as a vaccine component, or may be a therapeutic agent. The therapeutic agent may be a therapeutic polypeptide.

The target polypeptide may be derived from a pathogen. The pathogen may be selected from the group consisting of a virus, a bacterium, a parasite and a fungus. The virus may be an animal virus. The animal virus may be selected from the group consisting of an influenza virus, a respiratory syncytial virus (RSV), a human immunodeficiency virus (HIV), a hepatitis B virus (HBV), a hepatitis C virus (HCV), a human papillomavirus (HPV), an Ebola virus, a Yellow fever virus, a rotovirus, and a vesicular stomatitis virus (VSV).

The target polypeptide may be a native surface polypeptide. The native surface polypeptide may be a hemagglutinin of an influenza virus.

The target polypeptide may be an artificial surface polypeptide. The artificial surface polypeptide may be protective antigen 83 (PA83) from Bacillus anthracis, Pfs25 from Plasmodium falciparum or other soluble protein or peptide.

The influenza virus may be selected from the group consisting of an Influenza A virus and an Influenza B virus. The influenza virus may be selected from the group consisting of Influenza A Indonesia 05/05 strain, Influenza A virus California/04/2009 (H1N1) strain, Influenza A/Victoria/3/75 (H3N2) strain and Influenza B Hong Kong/330/2001.

The target polypeptide may be a therapeutic agent. The therapeutic agent may be a therapeutic polypeptide.

The transmembrane domain may be native or foreign to the surface exposed portion of the surface polypeptide. The transmembrane domain may be derived from a cellular or viral transmembrane protein. The viral transmembrane domain may be derived from the transmembrane protein from which the cytosolic tail is derived.

According to another aspect of the present invention, a method of producing virus-like particles (VLPs) in a plant cell, a plant, or a portion of a plant is provided. The method comprises introducing one or more nucleic acid molecules into the plant cell, the plant, or the portion of a plant. The one or more nucleic acid molecules comprise a first nucleotide sequence encoding a matrix protein derived from a first plant enveloped virus and a second nucleotide sequence encoding a surface polypeptide. The surface polypeptide comprises a surface exposed portion derived from a target polypeptide, a transmembrane domain, and a cytosolic tail. The cytosolic tail is derived from a transmembrane protein (e.g., glycoprotein) of a second plant enveloped virus. The first and second plant enveloped viruses may be the same or different, preferably the same. Either may be a plant rhabdovirus or non-rhabdovirus. Preferably, the first plant enveloped virus is a plant rhabdovirus. The method further comprises maintaining the plant cell, the plant, or the portion of a plant under conditions permitting co-expression of the matrix protein and the surface polypeptide such that the VLPs are produced. The VLPs may be substantially uniform in size. The method may further comprise purifying the VLPs from the plant cell, the plant or the portion of the plant.

The one or more nucleic acid molecules may be introduced into the plant cell, the plant or the portion of a plant by infiltration, particle bombardment, or inoculation. The one or more nucleic acid molecules may be introduced into the plant or a portion thereof transiently or stably.

Various immunogenic compositions are provided. In some embodiments, the immunogenic composition comprises an effective amount of the VLPs of the present invention, and the VLPs are substantially uniform in size. In other embodiments, the immunogenic composition comprises an effective amount of the VLPs produced by the method of the present invention, and the VLPs are substantially uniform in size. The immunogenic composition may further comprise an adjuvant and/or an excipient.

A method of inducing an immune response to the target polypeptide in a subject is provided. The method comprises administering to the subject an effective amount of the immunogenic composition of the present invention, wherein the VLPs are substantially uniform in size.

A method of inducing a protective immune response to a pathogen in a subject is also provided. The method comprises administering to the subject an effective amount of the immunogenic composition of the present invention, wherein the VLPs are substantially uniform in size. The target polypeptide is derived from the pathogen. The pathogen may be an influenza virus. The target polypeptide may be derived from a hemagglutinin.

A recombinant plant cell comprising one or more nucleic acid molecules is further provided. The one or more nucleic acid molecules comprise a first nucleotide sequence encoding a matrix protein derived from a first plant enveloped virus and a second nucleotide sequence encoding a surface polypeptide. The surface polypeptide comprises (a) a surface exposed portion derived from a target polypeptide, (b) a transmembrane domain, and (c) a cytosolic tail derived from a transmembrane protein (e.g., glycoprotein) of a second plant enveloped virus. The first and second plant enveloped viruses may be the same or different, preferably the same. Either may be a plant rhabdovirus or non-rhabdovirus. Preferably, the first plant enveloped virus is a plant rhabdovirus. Also provided is a plant or a portion thereof comprising the plant cell of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows (A) an electron microscopy image and (B) the organization of a plant rhabdovirus.

FIG. 2 is a diagram illustrating the genome organization of the T-DNA region of a miniBYV vector for co-expression of two target proteins, i.e., Target 1 and Target 2, each operatively linked to a regulatory region. L-Pro, papain like leader proteinase; Met, Hel and Pol, methyltransferase, RNA helicase, RNA-dependent RNA polymerase domains of the replicase, respectively; 2Enx35S, 35S promoter with dual enhancers from Cauliflower mosaic virus; NOS—Nopaline synthase terminator; LB and RB, left and right borders of the T-DNA, respectively.

FIG. 3 is a diagram illustrating the genome organization of the T-DNA region of a miniBYV-HAi-TM/M_(LNYV) or miniBYV-HAi-TM/M_(NCMV) vector for co-expression of an HAi-TM protein and a matrix protein of Lettuce Necrotic Yellows virus (LNYV) (M_(LNYV)) or Northern Cereal Mosaic virus (NCMV) (M_(NCMV)), respectively. L-Pro, papain like leader proteinase; Met, Hel and Pol, methyltransferase, RNA helicase, RNA-dependent RNA polymerase domains of the replicase, respectively; 2Enx35S; 35S promoter with dual enhancers from Cauliflower mosaic virus; NOS—Nopaline synthase terminator; LB and RB, left and right borders of the T-DNA, respectively.

FIG. 4 is a diagram illustrating the genome organization of the T-DNA region of a miniBYV-HAi-TM(G)/M_(LNYV) vector for co-expression of an HAi-TM(G)_(LNYN) protein and an M_(LNYV) protein. L-Pro, papain like leader proteinase; Met, Hel and Pol, methyltransferase, RNA helicase, RNA-dependent RNA polymerase domains of the replicase, respectively; 2Enx35S, 35S promoter with dual enhancers from Cauliflower mosaic virus; NOS—Nopaline synthase terminator; LB and RB, left and right borders of the T-DNA, respectively.

FIG. 5 depicts a representative western blot for time course analysis of HAi-TM expression detected by an anti-HAi 5G6 primary monoclonal antibody after infiltration. Reference HAi in the amount of 60, 30, or 15 ng was used as standard for quantification. Lanes 1 and 2, manual infiltrated N. benthamiana; 3 and 4, vacuum infiltrated N. benthamiana.

FIG. 6 shows (A) distribution of HAi-TM VLPs through a 10%-40% sucrose gradient and (B) morphology of the HAi-TM VLPs from fraction 10 of (A) detected by transmission electron microscopy (TEM) negative staining.

FIG. 7 shows (A) distribution of HAi-TM/M1 VLPs through a 10-40% sucrose gradient; and (B) morphology of the HAi-TM/M1 VLPs from fraction 10 of (A) detected by TEM negative staining.

FIG. 8 shows (A) expression of the influenza A/Indonesia/05/2005 matrix (M1) protein in leaf samples (lanes 1-3, different replicas from the infiltrated plants producing HAi-TM/M1 VLPs); and (B) absence of the M1 protein from the HAi-TM/M1 VLPs in the 10-40% sucrose gradient fractions as shown on FIG. 7A. Std, M1 protein standard.

FIG. 9 shows (A) distribution HAi-TM/M_(LNYV) VLPs in a 10-40% sucrose gradient and (B) immunogold labeling of the HAi-TM/M_(LNYV) VLPs with an anti-HAi 5G6 mouse monoclonal antibody.

FIG. 10 shows (A) distribution of HAi-TM(G)/M_(LNYV) VLPs through a 10%-40% sucrose gradient and (B) immunogold labeling of the HAi-TM(G)-M_(LNYV) VLPs from fraction 7 of (A) with an anti-HAi 5G6 mouse monoclonal antibody.

FIG. 11 shows the amino acid sequences of (A) a hemagglutinin of H5N1 avian influenza virus (A/Indonesia/05/2005) (HAi-TM) (SEQ ID NO: 1) having a PR1a Nicotiana tabacum signal peptide (underlined), an HAi ectodomain, an HAi transmembrane domain (bold), and an HAi cytosolic tail (italicized), and (B) a hemagglutinin of H5N1 avian influenza virus (A/Indonesia/05/2005) (HAi-TM(G)_(LNYV)) (SEQ ID NO: 2) having a PR1a N. tabacum signal peptide (underlined), an HAi ectodomain, a transmembrane domain of an LNYV glycoprotein (bold), and a cytosolic tail of an LNYV glycoprotein (italicized).

FIG. 12 shows the amino acid sequences of (A) a matrix protein of LNYV (M_(LNYV)) (SEQ ID NO: 3) and (B) a matrix protein of H5N1 avian influenza virus (A/Indonesia/05/2005) (M1 protein) (SEQ ID NO: 4).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that novel virus-like particles (VLPs) comprising a matrix protein derived from a plant enveloped virus and a surface polypeptide can be produced in plants. These VLPs are enveloped, and are substantially uniform in size, and may be used to design and manufacture effective human vaccines.

The term “protein” used herein refers to a biological molecule comprising amino acid residues. A protein may comprise one or more polypeptides. Each polypeptide may be a subunit of a protein. The protein may be in a native or modified form, and may exhibit a biological function when its polypeptide or polypeptides are properly folded or assembled.

The term “polypeptide” used herein refers to a polymer of amino acid residues with no limitation with respect to the minimum length of the polymer. Preferably, the polypeptide has at least 4 amino acids. A polypeptide may be a full-length protein, or a fragment or variant thereof.

The term “fragment” of a protein as used herein refers to a polypeptide having an amino acid sequence that is the same as a part, but not all, of the amino acid sequence of the protein. Preferably, a fragment is a functional fragment of a protein that retains the same function as the protein.

The term “variant” of a protein used herein refers to a polypeptide having an amino acid sequence that is the same as that of the protein except having at least one amino acid modified, for example, deleted, inserted, or replaced, respectively. The amino acid replacement may be a conservative amino acid substitution, preferably at a non-essential amino acid residue in the protein. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are known in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). A variant of a protein may have an amino acid sequence at least about 80%, 90%, 95%, or 99%, preferably at least about 90%, more preferably at least about 95%, identical to the amino acid sequence of the protein. Preferably, a variant is a functional variant of a protein that retains the same function as the protein.

The term “derived from” used herein refers to an origin or source, and may include naturally occurring, recombinant, unpurified or purified molecules. The molecules of the present invention may be derived from viral or non-viral molecules. A protein or polypeptide derived from an original protein or polypeptide may comprise the original protein or polypeptide, in part or in whole, and may be a fragment or variant of the original protein or polypeptide.

The term “native” used herein refers to a molecule (e.g., a protein or polypeptide) that is naturally occurring. The term “artificial” used herein refers to a molecule (e.g., a protein or polypeptide) that is not naturally occurring, but synthesized artificially, for example, recombinantly or chemically.

According to one aspect of the present invention, a virus-like particle (VLP) is provided. The VLP comprises a matrix protein and a surface polypeptide. The matrix protein is derived from a first plant enveloped virus, preferably plant rhabdovirus, more preferably, lettuce necrotic yellows virus. The surface polypeptide comprises a surface exposed portion derived from a target polypeptide, a transmembrane domain and a cytosolic tail. The cytosolic tail is derived from a transmembrane protein (e.g., glycoprotein) of a second plant enveloped virus. For example, the cytosolic tail may be derived from a rhabdoviral glycoprotein. The transmembrane domain and the cytosolic tail may be derived from the same or different plant enveloped viruses, preferably from the same plant enveloped virus. In one embodiment, the transmembrane domain is derived from a target protein while the cytosolic tail is derived from a rhabdovirus glycoprotein. In another embodiment, the transmembrane domain and the cytosolic tail are both derived from the same plant rhabdoviral glycoprotein. The matrix protein, the transmembrane domain and the cytosolic tail may be derived from the same plant enveloped virus.

The VLP may be produced in a plant cell or other cells. The plant cell may be a cell in a plant, a plant part (e.g., leaf, stem, root, floral tissue, seed or petiole), or a cell culture medium. The plant may be a whole growing plant. Preferably, the plant cell is in a plant leaf. The cell culture media may be any media suitable for growing plant cells, preferably in suspension. The plant cell is preferably suitable for expression of a VLP. For example, the plant cell may be in N. benthamiana leaves. Other suitable plants include Nicotiana clevelandii, Beta vulgaris, Spinacia oleracea, Brassica spp, Lactuca sativa, Pisum sativum, Nicotiana tabacum, Plantago lanceolata, Tetragonia tetragonioides, Montia perfoliata, Stellaria media, Medicago truncatula, and Chenopodium foliosum.

The VLP may comprise a membrane. The surface polypeptide of the VLP may be integrated into the membrane. The membrane may be derived from the cell in which the VLP is assembled, made or produced.

The matrix protein may be derived from a natural matrix protein of any plant enveloped virus. The matrix protein may have an amino acid sequence at least about 80%, 85%, 90%, 95%, or 99%, preferably at least about 90%, more preferably at least about 95%, further preferably at least about 99%, most preferably about 100%, identical to that of the corresponding natural matrix protein.

The surface exposed portion derived from a target polypeptide is a portion of the surface polypeptide that is on the surface of the VLP. The surface polypeptide may be an antigenic polypeptide, such as a vaccine component, or may be a therapeutic agent. The therapeutic agent may be a therapeutic polypeptide. The surface polypeptide, either in the VLP or purified from the VLP, may be used for various purposes. For example, the surface polypeptide may be antigenic and used to induce an immune response in a subject when introduced into the subject. It may also be used as a therapeutic agent to treat a disease or disorder in a subject when administered to the subject. It may further be used as a diagnostic agent for diagnosis of a disease or disorder in a subject when used in testing a sample from the subject suspected of having the disease or disorder.

The surface polypeptide may be derived from an antigenic target polypeptide, and capable of inducing an immune response in a subject when introduced into the subject, and may be used to form or become a vaccine candidate. It may comprise one or more epitopes (linear and/or conformational) capable of stimulating the immune system of a subject to make a humoral and/or cellular antigen-specific immune response. A humoral immune response refers to an immune response mediated by antibodies produced by B lymphocytes, or B cells, while a cellular immune response refers to an immune response mediated by T lymphocytes, or T cells, and/or other white blood cells. In general, a B-cell epitope contains at least about 5 amino acids but can be 3-4 amino acids, while a T-cell epitope includes at least about 7-9 amino acids and a helper T-cell epitope includes at least 12-20 amino acids. The target polypeptide may be derived from, in part or in whole, a natural protein (e.g., a surface protein or toxin subunit) of a pathogenic organism or pathogen. The target polypeptide may comprise an amino acid sequence at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%, preferably at least about 90%, more preferably at least about 95%, further preferably at least about 99%, most preferably about 100%, identical to that of the corresponding natural pathogenic protein. The pathogen may be an intracellular or extracellular pathogen. Exemplary pathogens include viruses, bacteria, parasites and fungi. The viruses may be animal or plant viruses, preferably animal viruses. An animal virus may be selected from the group consisting of an influenza virus, a respiratory syncytial virus (RSV), a human immunodeficiency virus (HIV), a hepatitis B virus (HBV), a hepatitis C virus (HCV), a human papillomavirus (HPV), an Ebola virus, a Yellow fever virus, a rotovarus, and a vesicular stomatitis virus (VSV). Preferably, the target polypeptide is derived from an influenza virus.

The target polypeptide may be a native surface polypeptide or an artificial surface polypeptide. The native surface polypeptide may be a hemagglutinin of an influenza virus. The artificial surface polypeptide may be protective antigen 83 (PA83), Pfs25, or other soluble protein or peptide. The PA83 is from Bacilus anthracis. The Pfs25 is from Plasmodium falciparum.

An influenza virus may be selected from the group consisting of an Influenza A virus and an Influenza B virus. The influenza virus may be selected from the group consisting of Influenza A Indonesia 05/05 strain, Influenza A virus California/04/2009 (H1N1) strain, Influenza A/Victoria/3/75 (H3N2) strain and Influenza B Hong Kong/330/2001.

The transmembrane domain of the surface polypeptide may be a native protein or an artificial protein. The transmembrane domain may be native or foreign to the surface exposed portion of the surface polypeptide or the target polypeptide. The transmembrane domain may be derived from a transmembrane protein, preferably the same transmembrane protein, from which the matrix protein and the cytosolic tail are derived. The transmembrane domain may be derived from a cellular or viral transmembrane protein, or a surface protein. The transmembrane domain may or may not be derived from the target polypeptide. The viral transmembrane protein may be a transmembrane protein from a plant virus. The plant virus may be a plant enveloped virus, preferably the plant enveloped virus from which the matrix protein and the cytosolic tail are derived. The transmembrane domain may be derived from a glycoprotein of a virus, preferably a glycoprotein of a plant enveloped virus, more preferably a glycoprotein of the plant enveloped virus from which the matrix protein and the cytosolic tail are derived.

A plant enveloped virus has a lipid envelope. This lipid envelope is typically derived from the membrane of a host cell from which the virus particle buds off. The plant enveloped virus may be a plant rhabdovirus or non-rhabdovirus. Examples of plant rhabdoviruses include Lettuce Necrotic Yellows virus (LNYV), Northern Cereal Mosaic virus (NCMV), Sonhus Virus (SonV) Broccoli necrotic yellows virus (BNYV), and other plant rhabdoviruses recognized by the International Committee on Taxonomy of Viruses (ICTV). Preferably, the plant enveloped virus is Lettuce Necrotic Yellows virus (LNYV).

LNYV and NCMV rhabdoviruses are characterized by monocistronic negative sense RNA and belong to the family Rhabdoviridae and the genus Cytorhabdoviruses. Plant rhabdoviruses are characterized by the bacilli-form structure (FIG. 1A) and are about 130-350 nm in length and about 40-100 nm in width (Jackson et al, 2005, Ann. Rev. Phytopathol. 43:623-660). The two proteins of the virus that are key for assembly of bacilli-form particles are matrix protein (M protein) and glycoprotein (G protein). M protein forms a layer inside the virion's envelope and interacts with the cytosolic tale of G protein (FIG. 1B).

According to another aspect of the present invention, a method for producing the VLPs of the present invention in a plant cell, a plant, or a portion of a plant is provided. The method comprises introducing one or more nucleic acid molecules into the plant cell, the plant, or the portion of a plant. The one or more nucleic acid molecules comprise a first nucleotide sequence encoding the matrix protein and a second nucleotide sequence encoding the surface polypeptide. The matrix protein is derived from a first plant enveloped virus. The surface polypeptide comprises a surface exposed portion derived from a target polypeptide, a transmembrane domain, and a cytosolic tail. The cytosolic tail is derived from a transmembrane protein (e.g., glycoprotein) of a second plant enveloped virus. For example, the cytosolic tail may be derived from a plant rhabdoviral glycoprotein. The transmembrane domain may or may not be derived from the target polypeptide. The first and the second plant enveloped viruses may be the same or different, preferably the same. Either may be a plant rhabdovirus or non-rhabdovirus. Preferably, the first enveloped plant virus is a plant rhabdovirus. The method further comprises maintaining the plant cell, the plant, or the portion of a plant under conditions permitting co-expression of the matrix protein and the surface polypeptide such that the VLPs are produced.

The VLPs produced by a method according to the present invention may be of any sizes. For example, the VLPs may have a diameter in the range of about 5-350 nm, including about 40-60 nm. Preferably, the VLPs are substantially uniform in size. The term “substantially uniform in size” used herein means that at least about 50%, 60%, 70%, 80%, 90%, 95% or 99%, preferably at least about 80%, more preferably at least about 90%, most preferably at least about 95%, of the VLPs have a diameter within less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%, preferably less than about 30%, more preferably less than about 20%, most preferably less than 10%, of the average diameter of all the VLPs. The diameter of a VLP may be the average or mean value of one or more measurements of the diameter of the VLP. The diameter of a VLP may be determined by conventional techniques known in the art, for example, by observation using an electron microscopy, sucrose gradient fractionation, size exclusion chromatography, and dynamic light scattering. The diameter values of the VLPs may be analyzed using various software, for example, ImageJ software (NIH, Bethesda, Md.) and other commercially available software.

The one or more nucleic acid molecules may further comprise a regulatory region operatively linked to the first or second nucleotide sequence. The regulatory region may be activated in a plant cell for the expression of the protein encoded by the first or the second nucleotide sequence. The regulatory region may include a promoter, for example, a plant viral promoter. Preferably, the first and second nucleotide sequences are in the same nucleic acid molecule, but linked to separate regulatory regions. The one or more nucleic acid molecules may further comprise a third nucleotide sequence encoding an additional surface polypeptide.

For each nucleic acid molecule, a vector comprising the nucleotide sequence of the nucleic acid molecule is provided. The vector may include a border sequence of a bacterial transfer DNA at either end, situated in a bacterial transfer DNA, to allow for delivery of the nucleic acid into a plant cell. Specifically, the vector may comprise one or more nucleic acid sequences derived from a Ti plasmid of a binary vector, and may also comprise a plant viral sequence. Such a vector, including elements of a Ti plasmid and a viral vector, is also called a launch vector. This vector may also be used for co-expression of the protein or polypeptide of interest (e.g., the matrix protein and/or the surface polypeptide) with a protein such as a silencing suppressor or a post-translational modifying enzyme such as PNGaseF, to modify, affect expression and/or increase production of the protein of interest by, for example, facilitating maturation or accumulation of the protein.

Each nucleic acid molecule may be introduced into the plant cell, the plant, or the portion of a plant (e.g., leaf, stem, root, floral tissue, seed or petiole) using techniques known in the art. For example, the nucleic acid molecule may be delivered via infiltration, particle bombardment, or inoculation. The nucleic acid molecule could be used as a part of an inducible system activated by, for example, chemical, light or heat shock. Preferably, the nucleic acid molecule is introduced into the plant cell via infiltration. The nucleic acid molecule may be introduced transiently or stably.

A recombinant plant cell comprising one or more nucleic acid molecules is provided. The one or more nucleic acid molecules comprise a first nucleotide sequence encoding a matrix protein and a second nucleotide sequence encoding a surface polypeptide. Preferably, the first and second nucleotide sequences are in one nucleic acid molecule. The matrix protein is derived from a first plant enveloped virus. The surface polypeptide comprises a surface exposed portion derived from a target polypeptide, a transmembrane domain and a cytosolic tail. The cytosolic tail is derived from a transmembrane protein (e.g., glycoprotein) of a second plant enveloped virus. The first and second plant enveloped viruses may be the same or different, preferably the same. Either may be a plant rhabdovirus or non-rhabdovirus. Preferably, the first plant enveloped virus is a plant rhabdovirus. The transmembrane domain may or may not be derived from the target polypeptide. The cytosolic tail may be derived from a glycoprotein of a plant enveloped virus. For example, the cytosolic tail may be derived from a rhabdoviral glycoprotein. The first or second nucleotide sequence may be operatively linked to a promoter capable of being activated in the plant cell for co-expression of the matrix protein or the surface polypeptide, respectively. Also provided is a plant or a portion thereof comprising the plant cell of the present invention.

For production of the VLPs of the present invention, a plant cell, or a plant or a portion thereof may be maintained under conditions permitting co-expression of the matrix protein and the surface polypeptide, resulting in the production of the VLPs. Such conditions include suitable temperature, humidity, pressure, light/dark cycle, and illumination. Preferably, the matrix protein and the surface polypeptide are co-expressed in the same plant cells.

The production method may further comprise purifying the VLPs. Conventional purification techniques known in the art may be used. For example, the VLP may be purified from the plant cell using an antibody or a receptor capable of binding the surface polypeptide. The purification process may comprise extraction of the VLPs from the plant cell using an extraction buffer. After low speed centrifugation, supernatant may be clarified by filtration and used for chromatography. The purified VLPs may be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%, preferably at least about 50%, more preferably at least about 75%, most preferably at least about 95%, pure. The VLPs in a crude plant extract may be used.

An immunogenic composition is provided. The composition comprises an effective amount of the VLPs of the present invention. Preferably, the VLPs are substantially uniform in size. The effective amount of the VLPs is an amount of the VLPs sufficient to make the composition immunogenic, i.e., capable of inducing an immune response in a subject when introduced into the subject. The composition may further comprise an adjuvant and/or an excipient. The immunogenic compositions may be used to induce an immune response in a subject. Several methods of inducing an immune response are provided.

A method of inducing an immune response to the target polypeptide in a subject is provided. The method comprises administering to the subject an effective amount of the immunogenic composition of the present invention. Preferably, the VLPs are substantially uniform in size. The immunogenic composition may be administered in an appropriate number of doses.

A method of inducing a protective immune response to a pathogen in a subject is also provided. The method comprises administering to the subject an effective amount of the immunogenic composition of the present invention. Preferably, the VLPs are substantially uniform in size. The target polypeptide is derived from the pathogen. For example, the target polypeptide may be a hemagglutinin and the pathogen is an influenza virus.

The subject may be an animal, including a mammal, for example, a human, a mouse, a cow, a horse, a chicken, a dog, a cat, and a rabbit. The animal may be an agricultural animal (e.g., horse, cow and chicken) or a pet (e.g., dog and cat). The subject is preferably a human or a mouse, more preferably a human. The subject may be a male or female. The subject may also be a newborn, a child or an adult. The subject may have suffered or be predisposed to a disease or medical condition, which may be caused by or associated with a pathogen.

The immunogenic composition may be formulated, for example, for oral, sublingual, intranasal, intraocular, rectal, transdermal, mucosal, topical or parenteral administration. Parenteral administration may include intradermal, subcutaneous (s.c., s.q., sub-Q, Hypo), intranasal, intramuscular (i.m.), intravenous (i.v.), intraperitoneal (i.p.), intra-arterial, intramedulary, intracardiac, intra-articular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluids) administration. Any device suitable for parenteral injection or infusion of the composition may be used for such administration.

The term “an effective amount” refers to an amount sufficient to achieve a stated goal, and may vary depending on the stated goal and other factors. For example, an effective amount of VLPs in an immunogenic composition or an effective amount of an immunogenic composition comprising VLPs may vary depending on the physical characteristics of the subject, the nature and severity of the need of the VLPs, the existence of related or unrelated medical conditions, the nature of the VLPs, the means of administering the VLPs to the subject, and the administration route. A specific dose for a given subject may generally be set by the judgment of a physician. The immunogenic composition may be administered to the subject in one or multiple doses.

Various medicaments comprising the VLPs of the present invention are provided. The VLPs are preferably substantially uniform in size. The medicaments may comprise suitable adjuvants. In some embodiments, the medicaments are useful for inducing an immune response to the target polypeptide in a subject, and comprise an effective amount of the VLPs of the present invention. In some other embodiments, the medicaments are useful for inducing a protective immune response to a pathogen in a subject, and comprise an effective amount of the VLPs of the present invention, wherein the target polypeptide is derived from the pathogen. For example, the target polypeptide may be a hemagglutinin and the pathogen is an influenza virus.

For each medicament of the present invention, a method for preparing the medicament is provided. The preparation method comprises admixing the VLPs of the present invention with a pharmaceutically acceptable carrier and/or excipient. The VLPs are preferably substantially uniform in size.

The term “about” as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate.

Example 1 Construction of Mini-BYV Vectors

To demonstrate the feasibility of assembling VLPs using matrix proteins (M proteins) from different plant enveloped viruses, a rhabdoviral M protein and a target antigen containing a transmembrane (TM) domain and a cytosolic tail of a LNYV glycoprotein (G protein) were engineered for co-expression in plant cells. For co-expression of these proteins, a miniBYV vector was used (FIG. 2). The miniBYV vector contains a minireplicon derived from a Closteroveridae virus (e.g., Beet yellows virus), which comprises a nucleic acid sequence encoding only proteins required for replication of the Closteroveridae virus. Specifically, the M protein from LNYV or NCMV was co-expressed with a hemagglutinin (HAi) from H5N1 avian influenza virus (A/Indonesia/05/2005).

In brief, a gene encoding a HAi protein having its native TM domain (HAi-TM) was cloned into a miniBYV vector to generate the miniBYV-HAi-TM plasmid. The HAi-TM protein (SEQ ID NO: 1) included a PR1a Nicotiana tabacum signal peptide (underlined), an HAi ectodomain, an HAi native transmembrane domain (bold), and an HAi native cytosolic tail (italicized) (FIG. 11A). Subsequently, a gene encoding the M protein of LNYV (M_(LNYV) protein) or NCMV (M_(NCMV) protein) was introduced into the miniBYV-HAi-TM plasmid under the control of the closteroviral heterologous coat protein (CP) promoter to generate a miniBYV-Hai-TM/M_(LNYV) or miniBYV-Hai-TM/M_(NCMV) plasmid (FIG. 3). For VLP evaluation, positive clones were transformed into Agrobacterium tumefaciens strain GV3101 and then introduced into Nicotiana benthamiana plants to produce VLPs.

In parallel, the HAi gene was modified to encode a recombinant a hemagglutinin protein (HAi-TM(G)), in which the native TM and the cytosolic tail of HAi were replaced with the transmembrane domain and cytosolic tail from a glycoprotein of LNYV, respectively. A nucleotide sequence encoding the HAi-TM(G) protein (SEQ ID NO: 2), including a PR1a Nicotiana tabacum signal peptide (underlined), an HAi ectodomain, an LNYV glycoprotein transmembrane domain (bold), and an LNYV glycoprotein cytosolic tail (italicized) (FIG. 11B), was introduced into the miniBYV vector containing a nucleotide sequence encoding the M protein of LNYV (M_(LNYV) protein) (SEQ ID NO: 3) (FIG. 12A). The resulting construct, miniBYV-HAi-TM(G)/M_(LNYV) (FIG. 4), was sequenced, and positive clones were transformed into A. tumefaciens and then introduced into Nicotiana benthamiana plants to produce HAi-TM(G)-M_(LNYV) VLPs.

As a control, a gene encoding the matrix protein (M1 protein) of H5N1 avian influenza virus (A/Indonesia/05/2005) (SEQ ID NO: 4) (FIG. 12B) was introduced into the minBYV-HAi-TM plasmid under the control of the closteroviral heterologous coat protein (CP) promoter to generate the plasmid miniBYV-HAi-TM/M1. As described above, PR1a was used as a signal peptide. Positive clones were transformed into A. tumefaciens and then introduced into Nicotiana benthamiana plants to produce HAi-TM/M1 VLPs.

Example 2 Optimization of Infiltration Procedure to Express Hai

To evaluate the expression level of HAi and develop vacuum infiltration conditions, hydroponically grown N. benthamiana plants were used. To analyze the expression of HAi-TM, manual infiltration and vacuum infiltration of miniBYV-HAi-TM were performed using the same buffer. Five week old Nicotiana benthamiana plants grown in Rockwool in clam shells were used for vacuum and manual infiltration. Agrobacteria were grown in LB media, which was supplemented with 50 ng/ml Kanamycin and 50 ng/ml Hygromycin. One liter overnight cultures were grown at 28° C., shaking at 220 rpm for 18-24 hours.

Overnight concentration was determined by measuring the OD₆₀₀ for each culture. The agrobacteria were pelleted by spinning at 4000 g for 15 min at 4° C. Bacterial pellets were re-suspended in 100 ml of fresh MMA media (10 mM MgCl2; 10 mM MES, pH 5.85; and 20 μM acetosyringone). The final concentration for each culture was recorded after rocking for 2 hours at room temperature. Cultures containing miniBYV and P1/HcPro silencing suppressor (miniBYV requires use of a silencing suppressor to achieve good target expression levels) were mixed to ratios of OD₆₀₀, 1.0:0.2, respectively. For vacuum infiltration, clamshells were infiltrated with miniBYV:P1/HcPro containing agrobacteria. Infiltrated plants were fed 50 ppm hydrosol and placed in a growth room for 5-8 days.

For manual infiltration, 3-5 leaves of hydroponically grown Nicotiana benthamiana were manually infiltrated using a 10 cc syringe without a needle. Plants were fed with 50 ppm hydrosol and kept in the post infiltration room for 5-8 days.

HAi-TM expression levels were determined based on the amount of HAi-TM protein detected by an anti-HAi 5G6 monoclonal antibody in Western blot analysis (FIG. 5). HAi-TM expression was higher in vacuum infiltrated leaves at 6 days post infiltrations (dpi) and 7 dpi compared to manually infiltrated leaves (Table 1). For example, at 6 dpi, the HAi-TM protein was expressed at 249 mg/kg in vacuum infiltrated leaves, representing a 43% increase in expression compared to manually infiltrated leaves.

TABLE 1 Time course expresssion levels for HAi-TM Days Manual Post Infiltration Infiltration (mg/kg) Vacuum Infiltration (mg/kg) 5 73 46 6 174 249 7 120 123 8 180 105

Example 3 Plant-Produced HAi-TM VLPs

To characterize VLPs formation HAi-TM were cloned in to miniBYV vector using PacI/NheI restriction sites. The resulting plasmid miniBYV HAi-TM was transformed in to GV3101 Agrobacterium strain. Nicothiana benthamiana infiltrated leaves were harvested at 7 days post infiltration and VLPs were purified. Distribution of HAi-TM VLPs without any matrix protein through a 10%-40% sucrose gradient with peak fractions 9 and 10 was detected by an anti-HAi 5G6 mouse monoclonal antibody (developed by the Immunology group at Fraunhofer—USA Center for Molecular Biotechnology) (FIG. 6A). To evaluate the morphology of HAi-VLPs in fraction 10, VLPs from this fraction were analyzed by negative staining using transmission electron microscopy (TEM). HAi-TM VLPs with different sizes and shapes were observed (FIG. 6B).

HAi-TM/M1 VLPs having HAi protein with its native transmembrane domain and cytosolic tail coexpressed with M1 of influenza A/Indonesia/05/05 were produced by expressing miniBYV-HAi-TM/M1 in plants using vacuum infiltration as described in Example 2. HAi-TM/M1 VLPs were purified using a sucrose gradient, and characterized by Western blot analysis and electron microscopy using the anti-HAi 5G6 mouse monoclonal antibody, and were found to distribute through a 10%-40% sucrose gradient with peak fractions 9 and 10 (FIG. 7A). No M1 protein incorporation was detected in the same HAi-TM/M1 VLPs through the 10-40% sucrose gradient (FIG. 8A) using a polyclonal goat anti-M1 antibody from Meredian Life science, Inc, USA. Meanwhile, the expression of M1 protein was confirmed by western blot using a goat anti-M1 protein polyclonal antibody (FIG. 8B). No uniformed VLPs were detected after co-expression HAi-TM and Influenza matrix M1 protein (FIG. 7B).

Example 4 Plant-Produced HAi-TM/M_(LNYV) VLPs

HAi-TM/M_(LNYV) VLPs having HAi-TM protein (i.e., HAi protein with its native transmembrane domain and cytosolic tail) and LNYV M protein (M_(LNYV) protein) were produced by expressing miniBYV-HAi-TM/M_(LNYV) in plants using vacuum infiltration as described in Example 2. HAi-TM/M_(LNYV) VLPs were purified using a 10%-40% sucrose gradient and characterized by SDS-PAGE, Western blot analysis and electron microscopy. The presence of the HAi immunological determinant on the surface of these VLPs was confirmed by immunogold labeling using the anti-HAi 5G6 mouse monoclonal antibody (FIG. 9B). Also, this antibody was used to detect VLPs distribution in a 10-40% sucrose gradient with a peak in fraction 10 (FIG. 9A). The distribution of HAi-TM/M_(LNYV) VLPs was similar to that observed for HAi-TM/M1 VLPs (FIGS. 7A and 9A). There was no noticeable difference in the VLP morphology (e.g., shape and size) (FIGS. 7B and 9B).

Example 5 Plant-Produced Uniform HAi-TM(G)/M_(LNYV) VLPs

HAi-TM(G)/M_(LNYV) VLPs having HAi-TM(G) protein (i.e., HAi protein with a transmembrane domain and a cytosolic tail from a rhabdoviral glycoprotein (G) protein) and LNYV M protein (M_(LNYV) protein) were produced by expressing miniBYV-HAi-TM(G)/M_(LNYV) in plants using vacuum infiltration as described in Example 2. After separation of HAi-TM(G)/M_(LNYV) VLPs by a 10%-40% sucrose gradient, 1 mL fractions were collected and analyzed by Western blot analysis using the anti-HAi 5G6 monoclonal antibody. A shift in the target collection peak was observed. Unlike HAi-TM/M_(LNYV) VLPs, the HAi-TM(G)/M_(LNYV) VLPs were detected in fractions 6, 7 and 8 with a sharp peak in fraction 7 (FIG. 10A). Immunogold labeling following TEM showed round-shaped HAi-TM(G)/M_(LNYV) VLPs (FIG. 10B). The size of the HAi-TM(G)/M_(LNYV) VLPs determined by ImageJ software (NIH, Bethesda, Md.) was 50.5±15 nm. The HAi-TM(G)-M_(LNYV) VLPs were substantially uniform in size and shape.

All documents, books, manuals, papers, patents, published patent applications, guides, abstracts, and other references cited herein are incorporated by reference in their entirety. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims. 

What is claimed:
 1. A virus-like particle comprising a matrix protein from a first plant enveloped virus and a surface polypeptide, wherein the first plant enveloped virus is a plant rhabdovirus, and wherein the surface polypeptide comprises (a) a surface exposed portion from a target polypeptide wherein the target polypeptide is a polypeptide not from the first plant enveloped virus, (b) a transmembrane domain, and (c) a cytosolic tail from a transmembrane protein of a second plant enveloped virus.
 2. The virus-like particle of claim 1, wherein the first plant enveloped virus and the second plant enveloped virus are the same.
 3. The virus-like particle of claim 1, wherein the virus-like particle is produced in a plant cell, a plant, or a portion of a plant.
 4. The virus-like particle of claim 1, wherein the plant rhabdovirus is selected from the group consisting of Lettuce Necrotic Yellows virus (LNYV), Northern Cereal Mosaic virus (NCMV), Sonhus Virus (SonV), and Broccoli necrotic yellows virus (BNYV).
 5. The virus-like particle of claim 1, wherein the surface exposed portion of the surface polypeptide is antigenic.
 6. The virus-like particle of claim 1, wherein the target polypeptide is from a pathogen.
 7. The virus-like particle of claim 1, wherein the surface exposed portion of the surface polypeptide is therapeutic.
 8. The virus-like particle of claim 1, wherein the target polypeptide is a native polypeptide.
 9. The virus-like particle of claim 1, wherein the target polypeptide is an artificial polypeptide.
 10. The virus-like particle of claim 1, wherein the transmembrane domain is native or foreign to the surface exposed portion.
 11. The virus-like particle of claim 1, wherein the transmembrane domain is from the transmembrane protein.
 12. The virus-like particle of claim 1, wherein the transmembrane domain is from a cellular or viral transmembrane protein.
 13. A method of producing virus-like particles of claim 1 in a plant cell, a plant or a portion of a plant, comprising (a) introducing one or more nucleic acid molecules into the plant cell, the plant or the portion of a plant, wherein the one or more nucleic acid molecules comprise a first nucleotide sequence encoding a matrix protein and a second nucleotide sequence encoding a surface polypeptide and wherein the surface polypeptide comprises (i) a surface exposed portion from a target polypeptide, wherein the target polypeptide is a polypeptide not from the first plant enveloped virus, (ii) a transmembrane domain, and (iii) a cytosolic tail from a transmembrane protein of a second plant enveloped virus; and (b) maintaining the plant cell, the plant or the portion of a plant under conditions permitting co-expression of the matrix protein and the surface polypeptide, whereby the virus-like particles are produced.
 14. The method of claim 13, wherein the first plant enveloped virus and the second plant enveloped virus are the same.
 15. The method of claim 13, wherein the virus-like particles are substantially uniform in size.
 16. The method of claim 13, further comprising (c) purifying the virus-like particles.
 17. The method of claim 13, wherein the one or more nucleic acid molecules are introduced into the plant cell, the plant or the portion of a plant by infiltration, particle bombardment, or inoculation.
 18. The method of claim 13, wherein the one or more nucleic acid molecules are introduced into the plant cell, the plant or the portion of a plant transiently or stably.
 19. An immunogenic composition comprising an effective amount of the virus-like particles of claim 1, wherein the virus-like particles are substantially uniform in size.
 20. An immunogenic composition comprising an effective amount of the virus-like particles produced by the method of claim 13, wherein the virus-like particles are substantially uniform in size.
 21. The immunogenic composition of claim 19, further comprising an adjuvant or an excipient.
 22. A method of inducing an immunological response to a pathogen in a subject, comprising administering to the subject an effective amount of the virus like particles produced by the method of claim 13, wherein the target polypeptide is from the pathogen, and wherein the virus-like particles are substantially uniform in size. 